Synthesis, antibacterial, antibiofilm, and docking studies of chalcones against multidrug resistance pathogens

The escalating threat of drug-resistant microbes underscores the urgent need for novel antimicrobial agents. In response, considerable research effort has been directed towards developing innovative frameworks and strategies to address this challenge. Chalcones, known for their broad-spectrum biological activities, have emerged as promising candidates for combating drug resistance. In this study, a series of 2′-Hydroxychalcones (5a, 5b, 5c, and 5d) with varying electron withdrawing and donating groups were synthesized via Claisen Schmidt condensation. FT-IR, 1H NMR, and 13C NMR analyses were employed to confirm the structure of the synthesized compounds. Subsequent evaluation of the synthesized compounds revealed their potential as antibacterial and antibiofilm agents. Notably, compounds 5a and 5d exhibited potent antibacterial activity against multidrug-resistant (MDR) bacteria E. coli, P. aeruginosa, K. pneumoniae, and S. aureus, surpassing the reference drug Ciprofloxacin (30 μg/mL) and other synthesized compounds. Compound 5d showed a notable 19.5 mm zone of inhibition against K. pneumoniae. Furthermore, 5a (at a concentration of 30 μg) and 5d (at a concentration of 50 μg) exhibited statistically significant (P > 0.05) biofilm inhibition efficacy compared to Ciprofloxacin (30 μg/mL). The synthesized chalcones 5a-5d were also docked via PachDock molecular docking software for Glucosamine-6-phosphate (GlcN-6-P) synthase inhibition and showed that ligand 5a exhibited outstanding results with score 4238 and ACE value −160.89 kcal/mol, consistent with the observed antibacterial activity. These findings underscore the potential of chalcones, particularly 5a and 5d, as promising candidates for the development of new antimicrobial agents targeting drug-resistant microbes and biofilm formation.


Introduction
The perpetual battle against infectious diseases has been ongoing for humanity.In modern times, the treatment of these diseases necessitates the use of a multi-drug regimen for an extended duration.Multidrug resistance phenomenon has resulted in increased mortality, morbidity and higher healthcare costs.Unfortunately, this approach has resulted in the swift emergence of strains that are resistant to multiple drugs, as well as a significant problem of patient non-compliance [1].Over the past sixty years, antibiotics played a prominent role for the treatment of toxins produced by bacteria.In recent years, there is a huge rise in the range of bacteria that are harmful and resistant to antibiotics [2].The huge problem now days are multidrug resistance bacteria such as Vancomycin-Resistant Enterococci etc which cause numerus infections [3].The increased MDR bacterial response towards antibiotics ultimately results in more drastic situations in terms of difficult-to-treat biofilm-related infections.Biofilms are intricate, multicellular communities of bacteria within an extracellular matrix.These structures have been shown to be associated with a significant proportion, approximately 80 %, of all bacterial infections [4].Biofilm formation has been shown to promote a significant increase in antibiotic tolerance, reaching levels up to 1000 times higher than what is typically observed in planktonic bacteria (individual, free-floating bacteria).Additionally, the formation of biofilms has been connected to long-lasting infections in situations like lung pneumonia in individuals with cystic fibrosis, otitis media, chronic wounds that don't heal, and the contamination of medical implants.This association with biofilms can lead to ineffective treatment of these infections, posing a considerable challenge to healthcare interventions.In order to provide new effective therapeutic agents, vigorous efforts are therefore required, and the development of new synthetics for microbial targets is a major problem for medical chemistry [5].General clinical experience indicates that single target drugs, although having good inhibitory activity against a specific target, are not effective agents for the biological system [6].This methodology creates it impossible to define a unique cure and specifically or at certain stages of its synchronization targets the virion without destroying the host cells [7].
Certain substituted chalcones and their derivatives, along with some of their heterocyclic analogues, have been documented for their potent biological properties, which have been shown to be harmful to the growth of microbes, tubercle bacilli, malarial parasites, and intestinal worms.Furthermore, it offers a versatile foundation for the construction of heterocyclic scaffolds through combinatorial assembly during synthesis [23].
Many chalcones such as Licochalcone, Xanthongelol, Isobavachalcone and other chalcones derivative (Fig. 2) have been approved for clinical use which shown an excellent antimicrobial activity [24].Since the chalcone spine is very useful in the preparation of substances with medicinal potential for different diseases [25] Several chalcone derivative like Isobavachalcone was extracted from Psoralea corylifolia which demonstrated significant antimicrobial effect with MIC of 8 μg/mL on MRSA strains [26].Chalcone exhibited significant antimicrobial activity against both Staphylococcus aureus and Escherichia coli [27].In 2023, M. Yadav et al. synthesized a chalcone derivative which showed MIC of 0.012 mol/mL against P. aeruginosa.Furthermore, a molecular docking study indicated that the chalcones have the potential to act as antibacterial inhibitors [28].A. Asadzadeh et al. in 2023, synthesized chalcone derivatives showed a good binding affinity to Streptococcus mutans Sortase A [29].
Glucosamine-6-phosphate synthase (G-6-P synthase) is a pivotal enzyme for the synthesis of UDP-N-acetyl glucosamine.G-6-P synthase is crucial for the initial steps in hexosamine biosynthesis.One of the products catalyzed by this enzyme, N-acetyl glucosamine, holds significant importance in forming the peptidoglycan layer present in the cell walls of both bacteria and fungi.G-6-P synthase holds great potential as a promising target for the discovery of novel antimicrobial compounds.Evaluating these potential compounds for their preservative efficacy may lead to the identification of safer and more effective preservatives for various applications [30].
Chalcones are proved to be potential GlcN-6-P synthase inhibitors [31,32].Taking into consideration the therapeutic significance of chalcones and as part of our ongoing research efforts to develop biologically potent molecules [33,34].Present research work is based on hypothesis that tested compounds might have promising antibacterial activities as novel compound against MDR bacteria.This could potentially help mitigate healthcare challenges and offer promising prospects for effective treatment.

Chemistry
We monitored the advancement of the reaction by employing silica gel 60 F254 TLC plates and visualizing the spots under UV radiation.FT-IR spectra were recorded using Agilent Technologies 41,630.Additionally, 1 H NMR and 13 C NMR spectra were obtained using AVANCE AV-400 MHz and AVANCE AV-500 MHz, Bruker 125 MHz, respectively.

In vitro antibacterial activity
The synthesized compounds (5a-5d) were assessed for their antibacterial activities using the disc-diffusion method on Muller Hinton agar medium [36].This study utilized Multidrug resistant (MDR) bacteria; E. coli, P. aeruginosa, K. pneumoniae, and S. aureus) bacteria previously isolated from cancer patients at a tertiary care hospital in Lahore, Pakistan.For the preparation of stock solution of each test compound, 10 mg of the compound was dissolved in 10 mL of DMSO.Following this, different concentrations, ranging from 30, 50, 100, 200, 400, to 800 μg/mL were prepared by diluting the stock solution accordingly.The paper disks (5.0 mm) were soaked in each concentrations for 5 min were placed on the surface of the freshly swabbed bacterial cultures on swabbed nutrient agar plates.The antibiotic disk of Ciprofloxacin (30 μg/mL) was used as standard drug and negative control at same plate.Following this, the plates were placed in an incubator set at 37 • C for a duration of 24 h.The zones of bacterial growth inhibition by each compound were then measured in millimeters (mm).

Antibiofilm assay (microreader plate)
To assess the efficacy of synthesized chalcones in inhibiting biofilm formation by bacterial pathogens (E.coli, P. aeruginosa, K. pneumoniae, and S. aureus), we quantitatively analyzed biofilm formation using a microplate reader, following the method outlined by Qurashi & Sabri [37].Microreader plate is used to quantify biofilm formation.Microbial culture is prepared in Luria-Bertani (LB) Broth and adjusted to 0.5 at OD-600 nm to ensure equal cell density 10 8 CFU/mL 180 μl of bacterial culture was dispensed into each well of sterile polystyrene 96-well-flat bottom plates.Then, 20 μl of each of the eight chalcone derivatives with varying concentrations were added using DMSO as a solvent.(30, 50 μg/mL).The microplates were placed in the incubator at 37 • C for a duration of 92 h, a time previously determined to be optimal for these cultures, without agitation.Afterward absorbance of planktonic cells was recorded at 600 nm.Next, the liquid culture was removed, and each well was stained with 100 μL of 1 % aqueous crystal violet for 20 min at room temperature.After 20 min of staining of adhered cells alongside walls of plates, rinsing with sterile distilled water was done.After drying, 200 μL of 33 % acetic acid was added to each well to dissolve the adhered cells.Following this, the absorbance of the plates was measured at 570 nm using a microplate reader.The Biofilm pattern of adhered cells was quantified as Normalized value (OD 570 nm/OD 600 nm) by excluding the difference of planktonic cells.All assays were carried out in triplicates.Classification of bacterial adherence summarized in Graph, based on "ANOVA" with Fisher's least significant difference (LSD) values.

Results and discussion
Chalcones were synthesized through Claisen-Schmidt condensation reaction, This involved reacting 2-Hydroxy-5-nitroacetophenone (1) with different aromatic aldehydes in the presence of KOH (Scheme 1).The synthesis of chalcones resulted in satisfactory yields, ranging from 58 % to 72 %.To characterize the synthesized compounds, infrared (IR), 1 H NMR, and 13 C NMR techniques were employed.In the 1 H NMR data of compounds 5a-5d, the presence of 2ʹ-OH was confirmed by observing signals in the range of δ12.71 -δ12.95.Signals within the range of δ7.75 -δ7.93 indicated the presence of α-H and β-H.Notably, the observed coupling constant value of 15 Hz clearly pointed to the trans configuration of the chalcones.Furthermore, other aromatic protons were also detected in their respective regions during the analysis.Fig. 3 presents a summary of the physiochemical data of the synthesized compounds.

Antibacterial activity
Chalcones are known for their wide range of biological activities, which are affected by the substitutions attached to the chalcone ring.In this study, the antibiotic potential of the synthesized compounds was assessed individually using the Kirby Bauer disk diffusion method.The results were quantified by measuring the zone of inhibition (ZOI) for each compound, alongside positive and negative controls.Chalcone derivatives that displayed a ZOI greater than 6 mm were considered to have promising antimicrobial properties Scheme 1. Scheme for the synthesis of 2-hydroxy-5-nitro chalcone.
T. Nawaz et al. [39].Among the chalcone derivatives tested (5a-5d), all demonstrated in vitro antimicrobial activity against a panel of standard strains of Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E.coli, P. aeruginosa, and K. pneumoniae).Compounds 5a and 5d demonstrated the most significant antibacterial efficacy against the examined pathogens at a concentration of 50 μg/mL, exceeding both the reference drug Ciprofloxacin (30 μg/mL) and all other synthesized compounds.There was a general trend of higher growth inhibition in terms of zone development by isolates P. aeruginosa and S. aureus as compared to rest of bacterial isolates.These findings highlight the potential of compounds 5a and 5d as potent antibacterial agents.Compound 5a features an electron-donating -OH group at position 2 and an electron-withdrawing -NO 2 group at position 5 of the A ring.Additionally, on B ring, compound 5a features an electron-withdrawing fluoro group at position 4. Compound 5a (50 μg/mL) has highest antimicrobial efficacy against tested pathogens with 21 mm zone of inhibition (ZOI) against E. Coli, 28 mm against P. aeruginosa, 29 mm against S. Aureus and 21 mm against K. pneumoniae which is comparable to standard ciprofloxacin (Table 1).It was reported that chalcones containing 2,4-dichlorophenyl and 2,4-difluorophenyl rings proved to be potent antibacterial agents [38].Presence of fluoro group on either A or B ring of chalcones activate the compound for antibacterial activity [39,40].Lagu et al. have reported that the chalcones bearing trifluoromethoxy group have more effective antibacterial activity [41].Compound 5d has electron donating -OH group at position 2 and electron withdrawing -NO 2 group at position 5 of A ring.Additionally, B ring 5d has electron donating methoxy group at position 4, Compound 5d showed excellent antimicrobial efficacy with 22 mm zone of inhibition (ZOI) against E. Coli, 29 mm against P. aeruginosa, 28 mm against S. Aureus and 19.5 mm against K. pneumoniae which is comparable to standard ciprofloxacin (Table 1).So the compound 5a and 5d has showed comparable zone of inhibition as compared to other synthesized compounds as well as standard as shown in Table 1.Shaikh et al. have reported that the chalcones bearing methyl groups are significant antibacterial agents [42].
This information is supported by Konduru et al. (2013) who observed antibacterial activity of hybrids Halo-chalcone (F, Cl, Br) which showed significant efficacy against B. subtilis [43].The significant antimicrobial activity of the chalcone molecule might be due to the methoxy and hydroxyl groups present on ring A or ring B [44].These functional groups likely contribute to the molecule's ability to effectively combat microbial pathogens [45,46].In general, chalcone with groups (-OH and methoxy) statistically enhanced the antibacterial activity [47].The lipophilicity of ring A in the hydroxyl chalcones was significant for their antibacterial activity [48].At MIC 6.25 g/mL against the tested microorganisms, hydoxy and haloginated chalcone compound shown good antibacterial activity [49].Li et al. have shown that chalcones bearing hydroxyl groups are active antibacterial agents against gram positive bacteria [50].Syahri, J. et al. synthesized 2-hydroxy linked chalcones that exhibits significant antibacterial efficacy against different gram positive and gram negative bacteria [51].Cationic groups such as amino group on A or B ring of chalcones are responsible for antibacterial activity [52].

Antibiofilm activity
Biofilm is a type of bacterial association usually developed on abiotic and biotic surfaces or at interfaces between air and liquid, covered by a extracellular bacterial polysaccharides, different proteins and nucleic acids in which bacteria multiply.Microbial biofilmrelated infections are an essential part of both symptomatic and chronic bacterial diseases in both humans and animals and also cause food spoilage [53].Biofilm colonies offer significant advantages to bacteria in terms of protective creation, but they are notorious for their antibiotic resistance.Chronic biofouling, food degradation, water pollution, and toxic substances are all caused by bacterial biofilms [54].Most antimicrobials drugs are unable to penetrate the biofilm because to Efflux pump (EP), which acts as a secondary barrier to bacterial cells.It is the main reason of antibiotic resistance through combinatorial mechanism of drug and efflux pump inhibitors the antibiotic resistance could be reduced.Chalcones have been tested for EPI activity against S. aureus native pump NorA As a result, in biofilms chalcone have been found to be effective in degrading Efflux pump (EP) [55].Quorum sensing (QS) system plays a crucial role in coordinating the formation of biofilms, secretion of virulence factors, mobility, and development of drug resistance, thereby influencing crucial bacterial cellular processes.Chalcones are proved to be active agents against quorum sensing to lower down the biofilm formation [56].
Biofilm formation of bacterial pathogens was tested in the presence and absence of synthesized compounds.The current study demonstrates that chalcones is effective against harmful bacteria that form biofilms.The capacity of chalcones to remove biofilms and disperse them makes it particularly well suited to the treatment of invasive bacteria and illnesses [57].
Anti-biofilm activity of chalcone against pathogenic isolates showed consistent results.So the efficacy of the four synthesized chalcones on the inhibition of the biofilm shows significant results (P > 0.05) for pathogens.The highest biofilm formation was recorded by S. aureus and K. pneumoniae as compared to the rest of isolate treatment and controlled experiment in (Fig. 5).However, in planktonic stage, the growth of P. aeuroginosa was highest as compared to all other pathogens but the trend was reversed for adherence (Normalized biofilm).In Fig. 4, the biofilm assay demonstrated that compounds 5a (at a concentration of 30 μg) and 5d (at a concentration of 50 μg) exhibited the most effective inhibition against all tested pathogens as compared to the reference drug Ciprofloxacin (used at 30 μg/mL) and other synthesized compounds.The chalcone derivative 5a, featuring hydroxyl and nitro groups in the A ring and a fluorine group in the B ring, displayed the most potent biofilm inhibition against all tested pathogens.5b also with the hydroxyl and Nitro groups as A ring, but other side of ring chloro group as ring B, showed the good biofilm inhibitory activity against bacterial strains (E.coli, P. aeuroginosa, K. pneumoniae and S. aureus).5c showed antibiofilm activity against P. aeuroginosa due to hydroxyl group on ring B, but this compound not effective against other three bacteria.Compound 5d demonstrated notable efficacy in inhibiting pathogens (E.coli, P. aeruginosa, K. pneumoniae, and S. aureus) and produced highly significant results (P > 0.05) regarding biofilm inhibition.
The findings presented align with the research conducted by Nijampatnam et al. in 2016, which also demonstrated that chalcone derivatives hold promise in hindering the formation of biofilms.The study supports the notion that these compounds can be effective in inhibiting biofilm development and validates the antimicrobial properties observed in this current investigation.Hydroxyl group and methoxy group of chalcones prevent the formation of S. aureus biofilm and Slobodníkova L. et al. assessed (2016) phloretin also reduced the effectiveness of E. coli biofilm without preventing the growth of microbes [58,59].Liu, M. et al., also assessed that xanthohumol is prenylated prevent S. aureus permeability and biofilm formation.In an already established biofilm, it also inactivated bacteria, likely disrupting the cohesion of the bacterial cytoplasmic membrane [60].Chalcones without hydroxyl groups on B ring are typically more effective against bacterial membranes.Moreover strongly hydroxylated lipophilic groups on A ring chalcone could be more disruptive to the framework of the membrane [61].Satokata et al., 2022 developed few hydoxy-haloginated chalcone which showed good antibiofilm activities 95 % inhibited cell viability at doses between 0.78 and 100 g/mL [49].
So this information is showed that the hydroxyl and methoxy groups compound disrupting more biofilm.5a, 5b and 5d significantly reduced the biofilm formation by all pathogens (Fig. 5).

Molecular docking studies
To explore the specific outcomes of the structure-activity relationship (SAR) at the GlcN-6-P synthase, molecular docking Fig. 4. Antibiofilm efficacy of (5a-5d) against pathogens.
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experiments were performed at the receptor site.The molecular docking tool "Patch Dock" was utilized to dock ligands 5a-5d with the crystal structure of GlcN-6-P synthase (PDB ID: 1MOQ) from the Protein Data Bank (PDB), as it is known for generating good molecular shape complementarity based on shape complementarity principles [62,63].Among the different docking solutions, "solution 1″ was chosen because it contained the most critical residues surrounding the binding pockets.The docked structures were then analyzed using Discovery Studio 4.5 Visualizer.In Table 2, the scores and atomic contact energies (ACE) of the docked conformations are presented, along with details of the hydrogen bonds and hydrophobic interactions formed by each ligand within the binding pocket of the receptor protein.The manuscript further discusses the relative interactions of the synthesized chalcones 5a-5d and the reference standard Ciprofloxacin with GlcN-6-P synthase (PDB ID 1MOQ) protein.

Docking of GlcN-6-P synthase with ligand (5a-5d)
GlcN-6-P synthase serves as a target enzyme essential for the biosynthesis of various precursors involved in the formation of the cell wall in both bacteria and fungi [31,64].Population of bacteria could be stopped by inhibition of this enzyme by a target molecule [65].
Standard Ciprofloxacin has shown score 4424 and an ACE value − 227.85 kcal/mol.Five hydrogen bonds were observed among hotspot and standard Ciprofloxacin.Hydrogen bonding was observed between the hydroxyl group of Ser 303 and carboxylic acid group of standard Ciprofloxacin.The distance between these bonds is 2.28 Å.Another hydrogen bond was noticed between hydroxyl group of Ser 347 and carboxylic acid group of Ciprofloxacin (2.18 Å).Third and fourth hydrogen bonds were showed between hydroxy group of Thr 352 and two carbonyl oxygens of standard Ciprofloxacin with average distance of 1.77 and 2.91 Å. Fifth hydrogen bond was observed between amino group of Val 605 and fluoro group of Ciprofloxacin (3.35 Å).Hydrophobic interactions of chalcones (5a-5d) Fig. 5. Effect of (5a-5d) on growth of E. coli, P. aeuroginosa, K. pneumoniae and S. aureus.with Gln 408 , Gln 348 , Leu 346 , Glu 396 , Ala 404 , Thr 302 , Gly 301 , Ala 400 , Lys 603 , Val 399 , Leu 484 , Leu 601 , Ser 349 and Gly 350 were also observed (Table 2; Fig. 6).Preliminary evaluations of the docked complexes (5a-5d) revealed that nearly all ligands showed overlapping with phenyl rings and positioned within the binding pocket (Fig. 7I).Conformations of ligands (5a-5d) with high binding affinity for the GlcN-6-P synthase are presented in Figs.7-8.
Table 2 shows that ligand 5a, which had the highest inhibition, also had the highest score (4238; ACE = − 160.89 kcal/mol) among the docked complexes.This indicates a strong correlation between experimental and theoretical findings.The docked complex of ligand 5a exhibited four hydrogen bonds.One hydrogen bond has shown between amino group of Arg 472 and oxygen of nitro group directly attached with phenyl ring of ligand 5a (2.48 Å).Hydrogen bonding was noticed between hydroxyl group directly attached with phenyl ring ofTyr 576 and fluoro group directly attached with phenyl ring of ligand 5a (3.27 Å).Third hydrogen bond has shown between oxygen of carbonyl group of Asp 474 and hydroxyl group directly attached with phenyl ring of ligand 5a (2.39 Å).There is an amino group of Asn 522 and oxygen of carbonyl group of ligands 5a potentially forms a hydrogen bond with an average distance of 2.54 Å (Fig. 7A and B).These hydrogen bonds are crucial for the interaction between the ligand and GlcN-6-P synthase.Amino acids His 566 , Val 567 , Ala 572 , Glu 569 , Gln 475 , Gly 473 , Pro 521 and Ala 551 showed hydrophobic interactions with ligands (Fig. 8).Pi-alkyl interactions have been visualized between ligand 5a and Ala 520 , Arg 472 pocket amino acid residues.
Ligand 5b exhibited a score (3790; ACE = − 92.94 kcal/mol).Two hydrogen bonds were observed; one with NH and other with oxygen of carbonyl group of Val 567 and hydroxyl of carboxylic acid group of ligand 5b with average distances of 2.66 and 3.29 Å respectively.The terminal phenyl ring establishes hydrophobic connections with the hydrophobic side chains of key amino acids, including Glu475, Asp474, Gly473, Leu525, Asn522, Pro521, Ala551, Asp548, and Tyr576, within van der Waals interaction ranges (Table 2; Fig. 7C and D).Pi-alkyl interactions have been visualized among ligand 5b and Ala 520 , Ala 572 binding pocket amino acid residues.
As showed in Table 2; ligand 5c with the score (3896; ACE = -92.78kcal/mol) showed a strong similarity between experimental and theoretical studies.Docked complex of ligand 5c showed hydrogen bonding between Glu 569 and carbonyl group of ligand 5c (2.73 Å).This hydrogen bond is regarded as to play a crucial role in the interaction of the ligand with the GlcN-6-P synthase (Fig. 7E and F).There were numerous hydrophobic interactions of the ligand with the binding pocket amino acids residues Val 567 , Tyr 576 , Ala 572 , Gln 475 , Asp 474 , Gly 473 , Leu 525 , Asn 522 , Pro 521 , Ala 551 , Asp 548 and His 566 observed.Pi-alkyl interactions have been showed between ligand 5c and Ala 520 , Arg 472 pocket amino acid residues.
Ligand 5d illustrated a high score (3958; ACE = − 102.77 kcal/mol).Evaluation of the interactions with the GlcN-6-P synthase binding pocket depicted hydrogen bond as well as hydrophobic interactions (Fig. 7G and H).A hydrogen bond has shown between the hydrogen of hydroxyl group of Glu 569 and oxygen of carbonyl group attached to the ligand 5d (2.87 Å).The terminal phenyl ring establishes hydrophobic connections with the hydrophobic side chains of key amino acids including Ala 551 , Pro 521 , Asn 522 , Leu 525 , Gly 473 , Asp 474 , Gln 475 , Ala 572 , Tyr 576 , Val 567 and His 566 residues within van der Waals interaction ranges.Pi-alkyl interaction has shown between ligand 5d and Ala 520 , Arg 472 hotspot amino acid.However, ligand 5a exhibited higher score compared to other ligands which correspond with the antibacterial screening outcomes.The critical amino acids exhibited in the hotspot are Glu 475 , Asp 474 , Gly 473 , Leu 525 , Asn 522 , Pro 521 , Tyr 576 .This computational study closely aligns with the findings from antibacterial screening, affirming the significant impact of the molecular docking analysis.

Conclusions
A series of Chalcones (5a-5d) have been successfully synthesized by Claisen-Schmidt condensation reaction.Among all the  synthesized compounds, the chalcones (5a and 5d) exhibited excellent antimicrobial activity as compare to reference drug and other synthesis compounds.The chalcones 5c was proved to be less effective.Antibiofilm activity of (5a-5d) were tested against pathogens (E.coli, P. aeuroginosa, K. pneumoniae and S. aureus).The compound 5a, 5b and 5d showed excellent inhibitory efficacy while the compound 5a and 5d showed significant antibiofilm activity against all four pathogens as compared to reference Ciprofloxacin.All the chalcones (5a-5d) were also docked using PachDock molecular docking software and exhibited that ligand 5a showed excellent results with score 4238 and ACE value − 160.89 kcal/mol which is in concordance with the antibacterial activity outcomes.Results of Chalcones formulations as bacterial growth inhibition and as antibiofilm agents seems promising and open new avenues towards drug discovery, however, further investigations are still needed.Future implications of the present study will be helpful for the success of

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Calculated from docked pose by using the ligand contact tool of Patch Dock.b Hydrogen bond length calculated from docked pose by using Ligand contact tool of Patch Dock.T. Nawaz et al.

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Fig. 8 .
Fig. 8. Analyses of hydrogen bonds (left) and hydrophobic contacts (right) of ligand 5a.Blue color in the hydrophobic interactions identifies favorable structural elements (atoms and torsions) contributing to entire binding energy within the GlcN-6-P synthase (PDB ID: 1MOQ), pink one is unfavorable, and white one is neutral.